Structure-Based Design of a Cyclic Peptide Inhibitor of the SARS-CoV-2 Main Protease

This paper presents the design and study of a first-in-class cyclic peptide inhibitor against the SARS-CoV-2 main protease (Mpro). The cyclic peptide inhibitor is designed to mimic the conformation of a substrate at a C-terminal autolytic cleavage site of Mpro. Synthesis and evaluation of a first-generation cyclic peptide inhibitor reveals that the inhibitor is active against Mpro in vitro and is non-toxic toward human cells in culture. The initial hit described in this manuscript, UCI-1, lays the groundwork for the development of additional cyclic peptide inhibitors against Mpro with improved activities.


INTRODUCTION
Antiviral drugs are desperately needed to help combat the COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), as well as epidemics caused by other coronaviruses in the future. 1,2 Antiviral drugs that slow or halt viral replication can lead to a shortened time to recovery from COVID-19, offering the promise of improved mortality rates and alleviation of the tremendous strain experienced by hospitals during the COVID-19 pandemic. 3 The main protease (M pro or 3CL protease) is one of the best-characterized drug targets for coronaviruses. 4,5,6,7,8,9,10 The SARS-CoV-2 main protease is a member of a of class homologous cysteine proteases that are needed for viral replication in diseases such as Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). These viruses cleave the initially translated viral polyprotein into its component proteins. Cleavage generally occurs immediately after a Gln residue, and the Gln residue is typically preceded by a hydrophobic residue, most often Leu. The residue that follows the Gln is often a small amino acid such as Ser, Ala, or Asn. M pro autolytically cleaves itself from the polyprotein. 11 Inhibiting M pro activity slows or halts viral replication, offering the promise of improved clinical outcomes for COVID-19 and other coronavirus diseases. Furthermore, there are no known human proteases with similar cleavage specificity to M pro , suggesting that it should be possible to develop inhibitors that target M pro without off-target toxicity.
The SARS-CoV-2 M pro amino acid sequence is 96% identical to the SARS-CoV M pro amino sequence, and the three-dimensional structure of the SARS-CoV-2 M pro is highly similar to the structure of the SARS-CoV M pro . 12 Peptide-based inhibitors previously developed to target the SARS-CoV M pro have effectively been repurposed and modified to target the SARS-CoV-2 M pro -N3 from Jin et al., 13b from Zhang et al., and 11a and 11b from Dai et al. 12,13,14 These inhibitors effectively block SARS-CoV-2 replication in cell-based studies, making them promising antiviral drug candidates. While the M pro inhibitors N3, 13b, 11a, and 11b have shown promise against inhibiting SARS-CoV-2 replication, additional M pro inhibitors will most likely be needed for their improved properties or to be used in combination therapies. 15 In this paper, we describe the design and preliminary evaluation of UCI-1 (University of California, Irvine Coronavirus Inhibitor-1), a first-in-class cyclic peptide that inhibits the SARS-CoV-2 M pro ( Figure 1). UCI-1 is designed to mimic the conformation of a C-terminal autolytic cleavage site of the SARS-CoV M pro , a naturally occurring M pro substrate. UCI-1 contains amino acid side chains from the P2, P1, P1', and P2' positions of the M pro substrate that are designed to fill the S2, S1, S1', and S2' pockets of the M pro active site ( Figure 1A). In UCI-1, the carboxyterminus of the P2' residue is linked to the amino-terminus of the P2 residue with a [4-(2aminoethyl)phenyl]-acetic acid (AEPA) group, creating a cyclophane. The (2-aminoethyl)phenyl group of AEPA is designed to act as a surrogate for a phenylalanine side chain at position P3' and fill the S3' pocket. Evaluation of the first generation cyclic peptide inhibitor UCI-1 in an in vitro M pro inhibition assay reveals that UCI-1 is active against SARS-CoV-2 M pro at midmicromolar concentrations. LC/MS analysis indicates that UCI-1 resists cleavage by M pro , despite containing a scissile amide bond. Furthermore, UCI-1 is found to be non-toxic toward human embryonic kidney cells at concentrations that inhibit M pro . The following details the design, synthesis, and preliminary evaluation of UCI-1.

RESULTS AND DISCUSSION
Design of the cyclic peptide inhibitor. We designed the cyclic peptide inhibitor based on the crystal structure of an inactive SARS-CoV M pro (C145A) variant with a 10 amino-acid Cterminal extension corresponding to the C-terminal prosequence of M pro (PDB 5B6O) ( Figure   2). 16    We recognized that upon linking Phe309 and Phe305 as described above, Phe305 and Gln306 were poised to form a β-turn in which the carbonyl of AEPA hydrogen bonds with the amino group of Gly307. We envision that β-turn formation in the cyclic peptide inhibitor will promote rigidity of the cyclic scaffold. To introduce additional conformational rigidity to the macrocycle, we also mutated Gly307 to serine, which is the most common residue at the P1' position among the 11 known SARS-CoV-2 M pro cleavage sites. The resulting cyclic peptide inhibitor UCI-1 was then synthesized and further studied as described below.  Figure S1 shows the results of the results of the continuous kinetic assay of UCI-1.

Synthesis
Initial rates for MBP-M pro activity in the presence or absence of UCI-1 or peptide-1a were obtained by fitting the linear portions of the curves from the continuous kinetic assay to a straight line. UCI-1 is active against MBP-M pro with an IC 50 value of ~150 µM ( Figure 4A). In contrast, the linear control peptide-1a shows little or no inhibition at concentrations at or below 256 µM ( Figure 4B). There appears to be a slight reduction in rate of cleavage of the fluorogenic substrate upon addition of 256 µM peptide-1a. This reduction in rate may reflect either slight inhibition of MBP-M pro or that the linear peptide acts as a competitive substrate at high concentrations. These findings indicate that the cyclic structure of UCI-1 is critical for its activity.    (Figures 2 and 7A). In the global minimum energy conformation, the AEPA residue acts as a rigid spacer, with the Phe, Gln, Ser, and Lys forming a bridge. As we had envisioned, the Phe and Gln residues adopt a β-turn conformation, with Phe at the i+1 position and Gln at the i+2 position. The Phe side chain is well situated to fit in the S2 pocket, and the Gln side chain is well situated to fit in the S1 pocket. The Ser, Lys, and AEPA residues, in turn, are poised to occupy the S1', S2', and S3' pockets. The macrocyclic scaffold of the cyclic peptide inhibitor appears to be particularly rigid. In the conformational search, the peptide backbones of most of the conformers adopt the conformation described above, differing only in side chain geometry and the type of β-turn formed by Phe305 and Gln306 ( Figure 7B).
Docking of the lowest energy conformer of UCI-1 with the SARS-CoV-2 M pro (PDB 6YB7) using Autodock Vina reveals that the inhibitor binds the active site of M pro in the envisioned manner ( Figure 7C). 23  we elected to share our initial hit, UCI-1, in this preprint. We hope that doing so will encourage others to also begin thinking about cyclic peptide inhibitors as promising drug candidates. We are currently pursuing next generation analogs of UCI-1 predicted to exhibit enhanced activity against M pro , and will report our findings from these pursuits in due course. iv. washing with DMF (3x). After coupling of the last amino acid, the terminal Fmoc group was removed with 20% (v/v) piperidine in DMF. The resin was transferred from the coupling vessel to a Bio-Rad Poly-Prep chromatography column.
iBabel was used to convert the UCI-1 minimized structured PDB file into a PDBQT file prior to docking. Docking was performed using AutoDock Tools and AutoDock Vina. In AutoDock Tools, a grid was chosen to encompass the active site of SARS-CoV-2 M pro in the size of 25x25x25 Å and with the coordinates x = 9.250, y = -5.944, z = 18.944. SARS CoV-2 M pro was treated as a rigid receptor in these calculations. The lowest energy cluster, as determined by AutoDock Vina was chosen to represent the SARS-CoV-2 M pro UCI-1 interaction model in Figure 7C.